Antibodies are naturally occurring proteins produced by immune systems in order to fight infections and eliminate pathogenic factors. Antibodies exert their functions by binding protein or non-protein antigens and triggering a defensive response for eliminating them.
In recent years, an entire therapeutic approach (named passive immunotherapy or passive serotherapy) has been built on the antigen-binding features of antibodies directed against both human and non-human molecules. Passive immunotherapy consists of the administration of pharmaceutical compositions comprising therapeutic antibodies with a defined antigen specificity for a pathogenic molecule (a toxin, a protein, a virus, a parasite, or a cell, for example) to patients whose immune system is unable to produce them in the amounts and/or with the specificity required to block and/or eliminate the pathogen (Dunman P M and Nesin M, 2003; Keller M A and Stiehm E R, 2000).
This approach has been successfully introduced into clinical practice in the early 1980s, and since then the use of therapeutic antibodies has rapidly expanded the opportunities for the treatment of a wide variety of diseases, including infectious diseases, immune-mediated diseases and cancer, resulting in constant growth of the therapeutic monoclonal antibody sector (Chatenoud L, 2005; Pavlou A and Belsey M, 2005; Laffy E and Sodoyer R, 2005).
Therapeutic antibodies suitable for passive immunotherapy are those having homogeneous, well-defined specificity and activities. These properties can be determined most accurately and reliably for a monoclonal antibody (i.e. an antibody secreted by a single clone of antibody-secreting cells) rather than for a polyclonal antibody (i.e. a complex mixture of antibodies secreted by different clones of antibody-secreting cells).
Since the 1970s, different technologies have been developed to isolate, propagate, and maintain large sets of cell lines, each derived from a single monoclonal cell culture secreting a monoclonal antibody (mAb), to be tested, using the appropriate assays, for identifying those having the desired properties.
Two important technical issues are common to all of these methods:                a) How to provide the antibody in amounts sufficient for the functional assays that are required for identifying and characterizing the antibody before performing any in vivo experimentation;        b) How to guarantee that the therapeutic antibody is not recognized itself as an antigen by the patient's immune system, triggering the elimination of the therapeutic antibody and/or immune inflammatory reactions that may be dangerous to the patient.        
The first issue is related to the difficulty in propagating and maintaining natural antibody-secreting cells in culture in enough time to have the biological material to test. This inconvenience has been solved by either immortalizing and maintaining in culture the primary antibody-secreting cells in which the nucleic acids encoding the antibodies have been initially generated and expressed, or by using recombinant DNA techniques for isolating antibody-encoding nucleic acids from these cells and transferring them into immortalized cells, in which they can be expressed and maintained.
In the past, primary antibody-secreting cells have been immortalized in cell culture conditions either by fusing them with cells already immortalized (forming hybrid cells or hybridomas that can be more easily maintained), or by using agents (such as virus) that alter the cellular machinery of primary antibody-secreting cells in a way that the cells propagate almost indefinitely.
The problem of guaranteeing the patient's safety has been solved in the past either by making use of cells and nucleic acids of human origin for producing antibodies, or by modifying the genes encoding non-human antibodies, that have an immunogenic potential, with sequence of human origin, an “humanization” process performed using recombinant DNA technologies.
In conclusion, passive immunotherapy can confer an efficient and rapid protection against infections and other pathologies. However, each method to isolate, screen, and produce monoclonal antibodies fully compatible with treatment in humans suffers from a different type of drawback, as briefly reviewed below.
The hybridoma technology, first described by Kohler and Milstein (Kohler G and Milstein C, 1975), allowed the isolation of continuously growing clones of antibody-secreting cells after being fused to an appropriate immortalized cell type. Hybridomas have been derived from human antibody-secreting cells (Olsson L and Kaplan H, 1980), but the process to produce human hybridomas has not proved to be robust, due to the lack of suitable human myeloma or lymphoblastoid fusion partners, and to the instability of human/human homohybridomas and human/murine heterohybridomas.
The humanization of murine antibodies can be achieved by grafting the antigen-binding region of the murine monoclonal antibody onto the backbone of a human antibody molecule, producing a chimeric molecule, and by substituting specific murine residues with other human amino acids to reduce antigenicity through molecular approaches (Hwang W and Foote J, 2005; Carter P, 2006).
There are numerous “humanized” antibodies currently in use or in clinical trials. However, these antibodies still contain 5-10% murine (or non-fully human) protein sequences and may elicit an immune response that limits the therapeutic efficacy of these drugs. In addition, the humanization process is labor-intensive and sometimes results in changes to antibody binding.
Therefore, this method has been mostly used with antibody-secreting cells originated in rodents immunized with the relevant antigen. Given that sequences of murine origin can be immunogenic in humans, the resulting mAbs can elicit toxic human-anti-murine responses, having an impaired antibody-dependent cellular cytotoxicity, and/or be rapidly cleared from the body. Moreover, even variable-region-identical antibodies may present different functional and immunogenic properties (Torres M et al., 2005).
Main approaches for producing fully human monoclonal antibodies are based on the cloning and the expression of human immunoglobulin genes using recombinant DNA technologies.
In a first case, libraries of DNA sequences encoding antibody fragments, including antigen-binding regions, can be amplified from human tissues and inserted into bacterial phage, allowing the “display” of antigen-binding fragments on the surface of the phage and the subsequent screening. Monoclonal antibodies against human pathogens have been produced, starting from the large antibody repertoire derived from patients that was cloned and screened using phage display technologies (Mancini N et al., 2004).
However, as employed under most circumstances, these libraries may be ineffective for identifying therapeutic antibodies since the antibody genes are not selected as the immune system does in vivo, on one side, for eliminating sequences in the human antibody repertoire that may elicit an immune response, and, on the other side, for selecting antibody sequences resulting from affinity maturation. Thus, complex in vitro affinity maturation and other technologies allowing direct sequence alterations are sometimes needed to improve antibodies from such libraries (Hoet R et al., 2005).
In a second case, transgenic mice expressing human antibody genes can be immunised with antigens of interest to produce murine cells expressing fully human antibodies (Kellermann S and Green L, 2002). This methodology has an advantage over traditional phage display methodologies because the antibodies are selected in vivo and may contain an increased frequency of high affinity antibodies. However, the mouse immune system acting in the mouse environment may not generate human antibodies with the appropriate specificity for an effective therapeutic use.
Thus, the ideal therapeutic antibody for passive immunotherapy is a human monoclonal antibody that is derived from human immune cells that have matured in a human being. However, the selection and the production of such antibodies is a complex and time-consuming process since conventional methods for producing and isolating populations of viable, immortalized human cells that secrete antibodies in cell culture conditions are inefficient.
The development and proliferation processes of human B cells, leading to their antigen specificity and long-term responses in vivo, and means to study the process in vitro using cells obtained from the immune system have been extensively reviewed (Banchereau J and Rousset, F, 1992; Crotty S and Ahmed R, 2004; Carsetti R, 2004; McHeyzer-Williams L and McHeyzer-Williams M, 2005). However, the isolation of human B cells expressing mAbs of interest has been hampered by the technical inability to produce stable human antibody-secreting cell lines, even when relevant binding or neutralizing activities can be detected.
Many different populations of antibody-secreting cells can be isolated from human donors having specific profiles (e.g. naive, vaccinated, more or less recently infected and seropositive individuals) and from different tissues (e.g. blood, tonsils, spleen, lymph nodes) where B cells reside and exert their activities (Viau M and Zouali M, 2005).
The identification of human monoclonal antibodies requires the extensive screening of the populations of immortalized B cells, wherein each cell secretes a specific monoclonal antibody in sufficient amounts for its characterization in cell culture conditions (Cole S et al., 1984; James K and Bell G, 1987; Borrebaeck C, 1989). However, the technologies for the selection, activation, and immortalization of antibody-secreting cells are still suffering from technical problems (yield of antibody, immortalization efficiency, overrepresentation of certain isotypes, cell stability and growth), leading to an insufficient number of cells and secreted antibodies available for screening assays.
Given the difficulty in obtaining stable hybridomas from human antibody-secreting cells, one method that has been extensively used to produce and isolate human antibody-secreting cells is the immortalization of human B cells with Epstein Barr Virus (EBV), which is also known to induce polyclonal B cell activation and proliferation (Sugimoto M et al., 2004; Bishop G and Busch L K, 2002).
Antibody-secreting cells have been produced by EBV immortalization using different sources of human B cells such as the peripheral blood of healthy subjects preselected using a labelled antigen (Casali P et al. 1986), lymph nodes, spleen, or peripheral blood from patients (Yamaguchi H et al., 1987; Posner M et al., 1991; Raff H et al., 1988; Steenbakkers P et al., 1993; Steenbakkers P et al., 1994), tonsils (Evans L et al., 1988), or pleural fluids (Wallis R et al., 1989).
However, because of low transformability, low clonability, and the inherent instability and heterogeneity of EBV-infected human B cells, valuable antibody-secreting B cells are often lost during this procedure (Chan M et al., 1986; James K and Bell G, 1987), obliging an additional cell fusion procedure to be applied after EBV infection (Bron D et al., 1984; Yamaguchi H et al., 1987; Posner M et al., 1991). In fact, some authors concluded that the best method for producing stable, human IgG antibody-secreting human monoclonal cell cultures was based on the fusion of human lymphocytes with a myeloma cell line (Niedbala W and Stott D, 1998; Li J et al., 2006), despite the technical difficulties with human hybridomas discussed above.
Various attempts have been directed at improving the immortalization process, for example by combining different approaches (immortalization with oncogenic virus, transformation with oncogenes, mini-electrofusion, mouse-human heterofusion) in a single process (U.S. Pat. No. 4,997,764; Steenbakkers P et al., 1993; Dessain S K et al., 2004). Human monoclonal antibodies have been isolated from B cells that have been activated and immortalized (in the presence or in the absence of an antigen), and by combining various manipulations in cell culture (Borrebaeck C et al., 1988; Davenport C et al., 1992; Laroche-Traineau J et al., 1994; Morgenthaler N et al., 1996; Niedbala W and Kurpisz M, 1993; Mulder A et al., 1993; WO 91/09115; Hur D et al., 2005; Traggiai E et al., 2004; Tsuchiyama L et al., 1997; WO 04/076677; WO 88/01642; WO 90/02795; WO 96/40252; WO 02/46233).
In general, the literature on methods for isolating and immortalizing cells that secrete antibodies, especially of human origin, does not provide a clear understanding on how to design the whole process for obtaining the largest repertoire of immortalized antibody-secreting cells, starting from the purification of cells that express antibodies from biological samples up to the screening of the antibodies that are secreted in cell culture conditions.
It would be clearly advantageous to provide methods for establishing more optimized processes in which, by applying specific means and conditions in cell culture for improving selection and viability of the antibody-secreting cells in an antigen-independent manner (but having specific isotypes of interest), a high throughput analysis of the secreted antibodies can be performed on the largest possible population of immortalized antibody-secreting cells maintained in cell culture conditions. Such a process would also expedite methods making use of molecular approaches to clone antibody genes because the population of B cells from which the antibodies having an isotype of interest are cloned may be repeatedly analyzed for the detection of cells secreting antibodies with a desired activity and stored in a viable state for future analysis.